Glycosylation site occupancy heterogeneity in Chinese hamster ovary cell culture

Asparagine linked (N-linked) glycosylation is an important secondary modification of recombinant proteins. Oligosaccharide chains can significantly influence glycoprotein properties such as specific activity, solubility, thermal stability and clearance rate in the blood stream. Despite the importance of glycosylation, it is an inherently variable process, and not all potential glycosylation sites are occupied with oligosaccharide. Furthermore, glycosylation characteristics can change with time in batch and fed-batch cultures. To investigate the interaction between cellular metabolism and glycosylation site occupancy heterogeneity, we studied how central carbon metabolism influences the availability of the nucleotide sugars which serve as sugar donors in glycosylation. We were able to characterize metabolism and glycosylation through a series of continuous culture (chemostat) experiments with Chinese hamster ovary cells producing recombinant human gamma interferon. Nutrient uptake and byproduct formation data obtained in chemostats were used to solve material balances for a biochemical network model of central carbon metabolism. From data obtained in glucose limited chemostats, we found